A significant advance in contemporary medicine is the introduction of point of care (POC) devices (POCd) for rapid, portable diagnosis. POCd provide rapid turnaround which aids therapeutic decisions, quick dissemination of test results to patients, thereby reducing physician workload and increasing patient satisfaction, reduced paper work, simplified sample tracking, and reduced need for specialized technicians. Despite numerous technological advances in the field of biosensors and biomarkers, currently, POCd's performance is limited to blood tests for glucose and cholesterol levels, blood pressure monitors, pregnancy tests, urine dipstick tests, scales, thermometers, otoscopes, and ophthalmoscopes only. Among them the blood glucose monitor occupies the largest POCd diagnostic sector and is the only one vastly available and affordable, followed by the cardiac and congestive heart failure POCd which are far behind, but the next fastest growing segment. POCd tests administered as multiplex panels provide further significant benefits by allowing screening of several cardiac markers in parallel with several other infectious markers and drugs of abuse, simultaneously saving time and providing comprehensive data. Screens for various types of influenza would aid diagnosis compared to more limited tests on only single strains.
Currently there are no devices commercially available for personalized medicine and continuous blood monitoring diagnostics for early detection, prevention, and treatment of infectious, cerebrovascular and pulmonary diseases like sepsis, stoke, chest pain, or drugs of abuse. An essential clinical requirement is a POCd that detects the presence and monitors in real time the concentration of specific biomarkers in a patient's bloodstream, sweat, saliva or other bodily fluids. Many intensive care patients develop infections which are not detected quickly and often lead to sepsis or shock and ultimately a large mortality rate. POCd circumvent the lengthy processing hours and high costs accompanying conventional off-site laboratory assays being highly valuable for medical staff but also for first responders which need time-critical diagnoses.
These devices enable rapid diagnosis by first responders or medical staff for time-critical diagnoses, such as for indicating whether patients are presenting with cardiac symptoms. Tests have been developed for other indications, such as infectious diseases, drugs of abuse, cerebrovascular disease, that are intended to circumvent the lengthy processing hours and high costs accompanying conventional in-house laboratory assays. Current POC devices are single use only. While this is suitable for many applications, there is an unmet need for continuous monitoring devices.
There is a clinical need for a device that can monitor and detect the presence of infections in intensive care patients. Currently, many intensive care patients develop infections that are not detected quickly, often leading to sepsis or shock and resulting in a large mortality rate. There is a significant need for a device that can continuously track the concentration of specific protein markers in a patient's bloodstream that are indicative of an infection, for instance.
The field of biosensors holds the promise of satisfying these needs. A biosensor is a device capable of specifically detecting the presence of selected chemicals, biochemicals, biomolecules, pathogens, toxins, or explosives in a sample, by indirectly measuring its interaction with a target molecule present at the biosensor surface. The miniaturization, integration and redesign possibilities offered by biosensors and microfluidics compared to conventional laboratory assays suggest that biosensors will dramatically enhanced diagnostic capabilities and development of POCd.
Emerging applications of biosensors also include food, water testing, bio-defense and “white powder” detection, and veterinary testing. Some of these applications have unique needs such as the need for ultra-fast response time in conjunction with bio-defense measures, or very high sensitivity necessary in food or water testing, for example when detecting a very low number of E. coli colony-forming units. Typical water testing products use reagents that must be incubated in flasks for 18-24 hours or longer, and indicate pathogen presence by changing color of the media. While these products are very effective and sensitive, the 24-hour incubation time is problematic when the contaminated water is a public drinking water supply. A biosensor device that continuously monitors water contaminant would warn the authorities within minutes of actual contamination.
Bio-defense presents unique issues as governmental and military agencies search for ways to rapidly and interactively detect terrorist agents like anthrax, botulism, malaria, Ebola virus, ricin, and other potential agents. Expensive test kits are currently used by the US Postal Service that incorporate real-time PCR to amplify and analyze crude samples obtained from air or suspicious “white powder” on packages and envelopes.
In several illustrative examples, devices that are capable of detecting the presence of selected chemicals or biological substances include biosensors that interact directly with a sample molecule to provide a signal identifying the test molecule. Biosensors are often functionalized chemically to make them selective. The readout can be electrochemical, as is often the case for small molecules (e.g. glucose), or can utilize fluorescence or other optical techniques for molecules such as proteins or DNA.
The biosensors are classified as labeled when the detection involves the presence of fluorescent, radioactive tag or chemiluminescence, and the interaction and detection steps are completely independent or label-free when the interaction and detection are simultaneous. Based on the physical property that is modified during the molecular interaction, the label-free biosensors are classified as optical (when based on ellipsometry, interferometry, whispering gallery modes, total internal reflection, or surface plasmon resonance (SPR)), mechanical (when based on quartz micro-crystal, surface acoustic waves, or cantilever), electrochemical (when based on amperometry, calorimetry, potentiometry, or conductometry) or thermodynamical (based on isothermal titration calorimetry, or differential scanning calorimetry). Typical label-free biosensors can often operate in a continuous reading mode or can be used multiple times, which differs from conventional laboratory assays requiring bulk reagent handling, usually yielding only a one-time test result. Among the label-free biosensors, SPR sensors are a fast and simple technology widely used in life science research, drug discovery, toxicology, food, environmental and industrial testing, because it allows label-free, real-time quantitative monitoring of interactions between an analyte (e.g. protein, peptide, nucleic acid, polynucleotide or virus solution) and target molecules bound to a gold surface with the ability to specifically interact with that analyte.
Despite certain advantages over other technologies like mass spectroscopy or ELISA because it allows label-free real-time monitoring of molecular interactions, SPR suffers from certain disadvantages that have prevented its use in potentially much larger fields including bio-defense, forensics diagnostics, high-throughput drug discovery, or POCd for human and veterinary diagnostics.
SPR utilizes a phenomenon arising from the interaction of photons with a metal surface which embodies an oscillating charge-density (electron cloud). We can excite SPR on surfaces with many different textures: planar, grooved, waveguide-patterned or closed-packed spheres, using either incident electron beams or photons. To enhance this coupling, attenuated total reflection (ATR) through prism couplers, waveguides, or diffraction gratings can be used. Incident electron-beams are the oldest method. Waveguides and diffraction gratings date from the late 1950s. In the late 1960s Otto and Kretschmann independently discovered two more practical optical excitation configurations involving prism couplers which made the technique widely available.
This exciting condition is usually met at the interface between a dielectric (e.g. glass) and a metal (illustratively gold or silver). The charge density wave (the electron cloud) is driven into resonance with an electromagnetic wave (the incoming photons), and this coupling reaches a maxima at the interface and decays exponentially into both media. This coupling is a surface bound plasma wave (SPW) which cannot be excited directly by randomly incident photons at a planar metal-dielectric interface because the SPW propagates more slowly than photons in free space. On smooth planar surfaces (e.g. Otto and Kretschmann configurations), a SPW is a non-radiative, longitudinal, p-polarized, charge-density wave bound to the interface between a medium with real positive refractive index (a dielectric sample-medium) and a medium with a complex (negative real part) refractive index (an SPR-supporting metal).
An incident beam of p-polarized light (the electrical field perpendicular to the propagation vector and parallel to the incidence plane defined by the propagation vector and the vector normal to the interface) passes through the dielectric prism and falls on the metal film. By varying the angle of incidence of the incident photons we slow them down and bringing them into resonance with the SPW, thus reaching the resonance angle, when the evanescent wave in the prism excites the SPW (FIG. 1). The light's p-polarization and incidence at the resonance angle are both essential to SPW excitation through a prism coupler. Incident p-polarized light optimally excites the SPW, which is itself p-polarized. A resonance conditions exists because the parallel-to-interface component of the incident-photon wave-vector matches SPW-vector and the energy of the photons transfers to the SPW, extinguishing the reflected beam. Owing to the strong concentration of the electromagnetic field in the dielectric (an order of magnitude higher than that in typical evanescent field sensors using dielectric waveguides) the propagation constant of the SPW, and consequently the SPR realization, is very sensitive to variations in the optical properties of the dielectric adjacent to the metal layer supporting SPW, namely the refractive index of the dielectric media which may be determined by optically interrogating the SPW. The thickness of the region of sensitivity varies with the wavelength off the applied energy, but is typically about 500 nm for wavelengths in the visible light range. The refractive index is modified by the presence of materials or impurities at the surface. This is the fundamental effect that can be used to identify binding activity at the surface.
Only few metals are can provide the negative sign dielectric constant. They have a resonant mode at which the constituent electrons resonate when excited by electromagnetic radiation having the right wavelength. Gold, in particular, has a spectrum with a resonance at visible wavelengths around 510 nm. In the case of the attenuated total reflection in prism couplers, the evanescent wave is sensitive to changes in the refractive index at the metal surface in contact with the media within approximately 200-400 nm of the surface, enhanced by the presence of a surface plasmon wave (SPW) (FIG. 1). Interactions between a bound substrate and a sample can thus be probed, measuring small variations in the reflection angle at maximum SPR production.
This effect can be harnessed to study binding between molecules, such as between proteins, RNA and/or DNA, or between proteins and pathogens (e.g. viruses, bacteria, fungi, and the like). In an illustrative exampler a surface functionalized with a specific antibotic (e.g. target molecule) will probe selectively for one antigen (e.g. antigen A) and discriminate specific binding from non-specific binding with other antigens (e.g. antigen B). That is, antigen A will be detected, but the weaker interactions between the functionalized protein bound to the surface and another antigen, say antigen B, can be distinguished by the corresponding kinetic profile.
Most commercial SPR instruments comprise a sample injection/rinsing device, a sensor surface made of a thin layer of gold (˜50 nm) coated on a glass slide that is brought in contact with a refractive-index matching semispherical dielectric prism. The gold layer is functionalized with target molecules that specifically bind to the analyte of interest. These commercial instruments further comprise a polarized light source, a photo-detector array, and various collimations and filtering optics on a goniometric mount as sketched in FIG. 1.
Using a semispherical prism, the angle of incidence at the dielectric/air interface is the same as at the first air/dielectric interface where the ray from the light source enters the prism. At the critical incidence angle (surface plasmon resonance angle, ΦSPR) at which incident photons couples to the SPW in the metal film the reflectivity of the film decreases more than 90% creating an evanescent plasmon field which is localized at the metal surface away from the glass. The evanescent wave's properties depend on the optical properties of the medium (e.g. biomolecules) in contact with the free metal surface of the sensor. Subtle changes in the refractive index of the medium, such as those associated with molecular absorption onto the surface induce detectable changes in the ΦSPR. The goniometric mount then adjusts the detector position to find this new angle and thus measures the change in SPR angle which correlates to the interaction kinetics of the antigen in solution with the target molecules fixed on the surface.
These types of SPR devices have a number of inherent limitations involving sensitivity, sample-sensing size, instrument size, complexity, and cost. Existing commercial instruments require large, complex, and delicate moving parts in order to optimize the incident beam and detector positions. The goniometric mount is relatively big and very delicate. The light source itself must provide polarized light. Typical sensitivity limits are on the order of 10−6 refractive index units corresponding to an angular resolution of 0.1 millidegree which can detect targets with a mass distribution of 1 pg/mm2 of adsorbed molecule and a size of at least 200 Da, but is not sensitive enough to provide useful detection for bio-terrorism agents in concentrations of 0.01 parts per billion as required by certain government standards. The typical planar sensor footprint is in the range of a few mm2 ( 1/16th mm2 in the Biacore Flexichip and 2.2 mm2 in the Biacore 3000) which creates a technical constraint on the ability to miniaturize the classical SPR sensors. A larger sensor area means that more test fluid must be provided to flow over the planar sensor. Moreover, the constraints on accuracy also require more test fluid to provide sufficient molecules or microparticles to be detected. Because of an SPR sensor's macroscopic size, arrays of sensing elements for multiplexed analysis require sample volumes too large for most technologies used for analytical integration. All of these limitations of conventional planar sensors reduce the throughput capability of the sensors. Overall the complexity of classical SPR instruments matches to those of a tabletop spectrometer which implies a high cost, typically on the order of several hundred thousand dollars.